Antitermination of transcription increases the expression of genes that are located downstream of transcription termination sites. Antitermination is sometimes caused by a stable modification of RNA polymerase that prevents it from recognizing or responding to termination signals. Nascent transcripts encoded by the cis acting antitermination sites ("put" sites) of bacteriophage HK022 promote such a modification: an RNA polymerase molecule that transcribes a put site acquires an increased ability to transcribe through downstream transcription terminators. Put dependent antitermination has little or no terminator specificity: both factor dependent and intrinsic terminators are suppressed. Efficient readthrough depends on the structure of the put transcript and of a highly conserved, cysteine rich metal binding motif located in the largest subunit of RNA polymerase (the beta' subunit). No additional protein factor is required. This suggests that a direct interaction between nascent put RNA and a domain of the beta' subunit is a step in antitermination. Comparison of the two natural put sites, analysis of put mutations, and determination of the sensitivity of put RNA to cleavage with structure specific nucleases suggest that the secondary structure of the biologically active antiterminator consists of two hairpin stems that are separated by a single unpaired base. Internal loops and bulges in the second stem are important for activity. To analyze the interaction between put RNA and polymerase, we blocked transcript elongation after put was transcribed, and determined the sensitivity of the put transcript to digestion with ribonucleases. We also treated the blocked elongation complex with oligonucleotides complementary to different segments of put RNA, allowed elongation to resume, and measured the efficiency of readthrough of a downstream terminator. The results suggest that hairpin 2 of put RNA remains associated with the elongation complex, and this interaction persists and is functionally important for antitermination even after polymerase has proceeded past the put site. By contrast, hairpin 1 appears to be required at an earlier stage of the reaction and might promote association of hairpin 2 with polymerase. Certain mutations that change residues close to the metal binding motif of the beta' subunit or alter hairpin 1 of put RNA prevent antitermination and also prevent the functionally important interaction between hairpin 2 and polymerase. Mutations that replace the conserved cysteines of the metal binding motif lead to put independent antitermination, suggesting that these mutations mimic the effect of put RNA binding to the wild type enzyme.